Withania somnifera or Ashwagandha is a medicinal herb in Ayurveda. Though the extract and purified molecules (Withanolides) from this plant have been shown to have different pharmacological activities, their effect on bone formation has not been studied. Clinical and experimental research on various Proteasomal Inhibitors (PI's) revealed that inhibition of proteasome activity by PI's is critical for the activation of osteogenic transcription factors and bone metabolism (Garrett IR et al 2003, Yaccoby S et. al 2010). Further, preclinical studies on synthetic PI's like bortizomib (Bzb) show that it promotes osteoblastogenesis and suppresses osteoclastogenesis by regulating the ubiquitin proteasome dependent degradation of proteins and acts as an anti-myeloma agent (Giuliani N et. al 2007, Shapovalv Y et. al 2009, Pennisi et al 2009). However, no natural PI is reported to have enhanced osteogenic effect in vivo. Withania somnifera is one of the most ancient sought after herbs for the preparation of herbal formulations and dietary supplements (Mirjalili M H et al 2009, Gupta P et al 2013). In traditional Indian medicine or Ayurveda, the leaves and roots of Withania are prescribed to cure inflammation related disorders (Mirjalili M H et al 2009, Gupta P et al 2013). This plant has been studied extensively for its biologically active constituents, steroidal lactones and withanolides (Grover A et al 2010, Matsuda H et. al 2001). The health benefits of Ashwagandha are supported by clinical trials in case of inflammation and immune modulation and reduction of arthritis pain (Rasool M et. al 2006, Mohan R et. al 2004). Pharmacological studies suggest that major chemical constituents for medicinal activities are withanolides from this plant. To characterize the bioactive entities in Ashwagandha, several research groups investigated the chemical constituents by diverse analytical tools. The first compound of this group to be isolated was withaferin A (WFA), a highly oxygenated withanolide. It is one of the major and main biologically active constituent from this plant (Mirjalili M H et al 2009, Sangwan R S 2008 et al). Exploration of chemical structure reveals that aromatic ketone structure of WFA interacts with the hydroxyl group of the N-threonine of the β5 subunit and inhibits chymotrypsin-like activity of 20 S proteasome. In silico studies suggest that two conjugated ketone bonds in WFA make it more susceptible towards nucleophilic attack by β5 subunit of proteasome (Yang H et. al 2007, Grover A 2010). Both in vitro and in vivo evidences provide proteasomal chymotrypsin subunit as a novel molecular target of WFA. Despite these studies on pharmacological activities of WFA, their mechanism of action is not fully understood.
The present invention relates to Withanolides, the secondary metabolites present in several members of Solanaceae family, most prolifically in Withania species, particularly Withania somnifera (Sangwan et al. Current Science 461-465, 2004). Withanolides are C28-steroidal lactones of triterpene ancestory, based on an intact or rearranged ergostane frame and chemically named as 22-hydroxy ergostane-26-oic acid 26,22-lactone. They are built on ergostane skeleton through appropriate oxidations at C-22 and C-26 to form a δ-lactone ring. Chemically or ergostanically, they are nomenclatured as 4, 27-dihydroxy-5β, 6β-epoxy-1-oxoWitha-2,24-dienolide, 4β, 20β-dihydroxy-5β, 6β-epoxy-1-oxo-Witha-2,24-dienolide, 5α, 20β-dihydroxy-6,7-α-epoxy-1-oxoWitha-2, 24-dienolide and 5α, 17α-dihydroxy-6, 7-α-epoxy-1-oxo-Witha-2, 24-dienolide, respectively.
Proteasomal Inhibitors in Diseases
Studies show the role of proteasome and the potential uses of proteasomal inhibitors for the treatment of human diseases. Several natural and synthetic compounds that act as proteasome inhibitors have been reported (Garret I R et al 2003, Elliott P J et al 2001).
Proteasomes degrade damaged cellular proteins and various short lived regulatory proteins and govern a wide range of cellular functions. The expression of cell cycle stimulatory and inhibitory proteins has a major role in the development and progression of cancer. Proteasome inhibitors can stabilize many cell cycle inhibitory proteins and cause cell cycle arrest and apoptosis, thus limiting tumor development. It has been demonstrated that lactacystin (PI) significantly reduced angiogenesis suggesting that these compounds could be beneficial in disease states that rely on the formation of new blood vessels (Oikawa et. al 1998). PS-341 has been shown to limit the production of metastases and limit angiogenesis in several experimental cancer systems (Hideshima T et.al 2001, Elliott P J et al 2001). Thus, proteasome inhibitors show significant potential as anti-angiogenesis agents, influencing both inflammatory conditions and the development of cancer metastasis.
The proteasome is intimately linked to the production of the majority of the class I antigens. It is therefore conceivable that excessive inhibition of the proteasome might also increase the chance of viral infections. Schwartz O et al 1998 reported that replication of the HIV-1 virus could be limited by the degradative actions of the proteasome and that the proteasome inhibitor, MG-132 or lactacystin, enhanced the ability of the virus to replicate.
Through its regulation of NF-kappa B, the proteasome is central to the processing of many pro-inflammatory signals. Once released from its inhibitory complex through proteasome degradation of I kappa B, NF-kappa B induces the activation of numerous cytokines and cell adhesion molecules that orchestrate the inflammatory response (Alkalay I et al 1995).
The role of proteasome in bone formation appears to occur mainly through regulation factor Runx2/cbfa1 and additional components associated with bone morphogenetic proteins BMPs and Wnt signaling. These proteasomal effects on osteo-blastogenesis and on the production of osteo-clastogenic factors in osteogenic cells indirectly impact osteoclastogenesis but the proteasome is also directly involved in osteoclastogenesis because it controls important signaling pathways such as NFkB pathway, in osteoclast precursor cells (Yaccoby S 2010).
TABLE 1List of proteasomal inhibitors (PI) at different stages of developmentSourceActivityStatusReferenceNatural (Bacterial)PILactacystinStreptomycesIncreasesPreliminaryIto y et al, 2011Sp.activity ofPre-clinicalSmads (instagevitro)EpoxomicinActinomycetesIncreases bonePreliminaryMundy GRstrain no 996-volume andPre-clinicalet al, 200517bone formationstageratePSI—Increases bonePreliminaryMundy GRvolume andPre-clinicalet al, 2005bone formationstagerateSynthetic PIBortezomibsyntheticOsteogenic inFDA approvedN Giulianivivo and infor MMet al, 2008vitro bothtreatmentMG-132syntheticStimulate bonePreliminaryGarrett IR
Sangwan et al. (US/2005.0266100 A1) discloses a process with improved isolation yields of Withaferin A, one of the Withanolides of Withania somnifera. 